The present invention relates to the field of Hepatitis B virus (HBV) diagnosis. More particularly, the present invention relates to the field of HBV genotyping and/or determination of the presence of HBV mutants in test samples.
The present invention relates particularly to a method for the rapid and reliable detection of HBV mutants and/or genotypes occuring in a test sample using specific sets of probes optimized to function together in a reverse-hybridisation assay.
Hepatitis B virus is a small enveloped DNA virus of approximately 3200 bp long. Historically it has been characterized on the basis of immunological reaction of the HBsAg with sets of monoclonal antibodies. Isolates were described as a, indicating the common determinant for all different subtypes, followed by subtype-specific combinations: dw, dr, yw, or yr. The latter are mutually exlusive pairs of determinants, covering the HBsAg amino acids 122 (d=lys, y=arg) and 160 (w=lys, r=arg). Several subdeterminants for w exist and can be ascribed to the appeareance of certain amino acid variants at codon 127. More recently, a genetic classification has been proposed, based on molecular analysis of the virus. This kind of analysis showed that in total six different genotypes exist, indicated from A to F, with a maximum genetic divergence of 8% when comparing complete genomes (reviewed by Magnius and Norder, 1995).
The genetic variability of HBV might be clinically important. Indeed, the genome variability might include some mechanisms by which HBV avoids immune clearance, and hence induces chronic infection. An important protein marker in inducing immune tolerance, virus elimination, and chronic infection, is HBeAg. The expression of this protein is strictly controled both at the transcriptional and translational level (Li et al., 1993; Okamoto et al., 1990; Yuan et al., 1995; Sato et al., 1995). Therefore, in the natural course of HBV infection, a well characterized stage of the disease is indicated as HBe-negative chronic hepatitis B (reviewed by Hadziyannis S. J., 1995). This phase is mostly due to the appeareance of preCore translational stop codon mutations. The overal genetic variability determines the frequency and physical location on the viral genome where these translational stop-codon mutations appear. The transcriptional regulation was proposed to be the mechanism for genotype A (and possibly also F), whereas the translational control was more likely to be found in the other genotypes (Li et al.; 1993; Sato et al., 1995). Contradictory to the translational regulation, it was shown that the transcriptional regulation was unable to block the HBeAg expression completely and was therefore proposed to categorize the phenotype of this mutant as HBe-suppressed, rather than as HBe-negative (Takahashi et al., 1995). In any case, these preCore mutants would lead to a destruction of the preexisting balance between HBeAg in circulation and the HBc-derived peptides presented by class I HLA molecules on the surface of infected hepatocytes, thereby diminishing the supressive effect of HBeAg on T cells, finally resulting in partial liberation of core-specific CTLs and leading to apoptosis of the infected hepatocytes. In general, after the emergence of the HBe-minus variants, the course of the viral infection is characterized by the progression of chronic hepatitis, which may lead to the development of cirrhosis and hepatocellular carcinoma (Hadziyannis, 1995).
Another issue for which the genetic variability or genotyping of the virus might be of relevance is in the development of vaccines where the response may be mediated by the virus type. Protection against HBV infection of all subtypes is conferred by antibodies to the common xe2x80x98axe2x80x99 determinant of the HB surface antigen (HBsAg). It has been shown that this xe2x80x98axe2x80x99 determinant presents a number of epitopes, and that its tertiary structure is most important for its antigenicity. The most important region lies between amino acid 124 and 147, but can be extended from amino acid 114 to 150. An adequate anti-HBs response, built up after vaccination, is in principle fully protective infection with a HBV strain harboring mutations in the xe2x80x98axe2x80x99 determinant region might result in vaccine failure, because the vaccine-induced humoral immune response does not recognize the mutant HBsAg. The most common vaccine-associated escape mutants are the substitutions of a glycine at position 145 to an arginine (G145R), K141E, and T126N. But a 2-aa insertion between aa position 122 and 123, and 8-aa insertion between aa 123 and 124 have also been found (Carman et al., 1990, 1995; Crawford, 1990; Waters et al., 1992).
Lamivudine is a (xe2x88x92) enantiomer of 3xe2x80x2 thiacytidine, a 2xe2x80x23xe2x80x2-dideoxynucleoside analogue, and is known to be a potent inhibitor of HBV replication through inhibition of the reverse transcriptase (RT) activity of the HBV polymerase. Lamivudine treatment can result in histological improvements in chronic hepatitis patients, and when given pre- and post-liver transplantation, it can prevent graft reinfection (Honkoop et al., 1995; Naoumov et al., 1995). However, after treatment, a hepatitis flare-up can be observed in most patients, with ALT elevations and HBV DNA that becomes detectable again. This HBV DNA rebound is associated with a new quasi species equilibrium. In a few cases, virus breakthrough during therapy was observed, due to the selection of lamivudine resistent HBV strains. The exact nature of this breakthrough has been ascribed to the accumulation of mutations in the RT part of the Polymerase. A similar mechanism in the HIV RT polymerase has been found, where upon lamivudine treatment, mutations accumulate in the YMDD motif (Gao et al., 1993). This YMDD motif is also present in the RT part of the HBV polymerase, and lamivudine-selected mutations in HBV have been found in this region (Tipples et al., 1996), as well as in other regions of the RT part of the polymerase (Ling et al., 1996). Penciclovir is another drug that has been shown to inhibit the reverse transcriptase activity of the HBV polymerase (Shaw et al., 1996), and mutations in the HBV polymerase may also be detected upon treatment with this drug.
From all this it can be concluded that the information on the following issues is essential for proper in vitro diagnosis, monitoring and follow-up of HBV infections:
genotype;
preCore mutations;
vaccine escape mutations;
RT gene mutations selected by treatment with drugs such as lamivudune and penciclovir.
To obtain all this information using existing technologies is compilcated, time-consuming, and requires highly-skilled and experienced personel.
It is thus an aim of the present invention to develop a rapid and reliable detection method for determination of the presence or absence of one or more HBV genotypes possibly present in a biological sample.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of the presence or absence of one or more variations in the HBV preS1 region and/or in the HBsAg region representing one or more HBV genotypes possibly present in a biological sample in one single experiment.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of the presence or absence of one or more HBV mutants possibly present in a biological sample in one single experiment.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of one or more mutations in the preCore region of HBV possibly present in a biological sample in one single experiment.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of one or more mutations in the HBsAg region of HBV possibly present in a biological sample in one single experiment.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for determination of one or more mutations in the polymerase (pol) gene region of HBV possibly present in a biological sample in one single experiment.
More particularly, it is an aim of the present invention to develop a rapid and reliable detection method for the simultaneous determination of one Dr several HBV Genotypes in combination with one or several HBV mutants possibly present in a biological sample in one single experiment.
It is also an aim of the present invention to provide a genotyping assay or method which allows to infer the nucleotide sequence at codons of interest and/or the HBV mutants of interest, and/or infer the HBV genotype possibly present in a biological sample.
Even more particularly it is also an aim of the present invention to provide a genotyping assay allowing the detection of the different HBV mutants and genotypes in one single experimental setup.
It is another aim of the present invention to select particular probes able to discriminate one or more HBV mutations in one of the above mentioned regions of the HBV genome and/or able to discriminate one or more HBV genotypes.
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HBV from mutant HBV sequences.
It is also an aim of the present invention to select particular probes able to discriminate wild-type and polymorphic variants of HBV from mutant HBV sequences.
It is also an aim of the present invention to select particular probes able to discriminate HBV genotype sequences.
It is moreover an aim of the present invention to combine a set of selected probes able to genotype HBV and/or discriminate different HBV mutants possibly present in a biological sample, whereby all probes can be used under the same hybridisation and wash conditions.
It is also an aim of the present invention to select primers enabling the amplification of the gene fragment(s) determining the HBV genomic mutations or variations of interest as discussed above.
The present invention also aims at diagnostic kits comprising said probes useful for developing such a genotyping assay and/or assays for detecting, monitoring or following-up HBV infection and/or assays for detecting HBV mutations.
All the aims of the present invention have been met by the following specific embodiments.
As a solution to the above-mentioned problem that it is essential for proper diagnosis, monitoring and follow-up of HBV infection to have information on the genotype of HBV present, the present invention provides an elegant way to tackle problems of such complexity which involves residing to a reverse hybridization approach (particularly on Line Probe Assays strips, as described by Stuyver et al., 1993). Using this technology it is possible to conveniently obtain all essential data in one test run. To achieve this goal, a set of probes needs to be designed and assembled which can detect all relevant polymorphisms in the HBV gene regions of interest.
The present invention thus particularly relates to a method for determining the presence or absence of one or more HBV genotypes in a biological sample, comprising:
(i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample;
(ii) if need be amplifying the relevant part of a suitable HBV gene present in said sample with at least one suitable primer pair;
(iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two nucleotide probes hybridizing specifically to a HBV genotype specific target sequence chosen from FIG. 1; with said probes being applied to known locations on a solid support and with said probes being capable of hybridizing to polynucleic acids of step (i) or (ii) under the same hybridization and wash conditions or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T of said target sequence is replaced by U;
(iv) detecting the hybrids formed in step (iii);
(v) inferring the HBV genotype present in said sample from the differential hybridization signal(s) obtained in step (iv).
The genotype specific target sequences can be any nucieotide variation appearing upon alignment of the different HBV genomes that permits classification of a certain HBV isolate as a certain genotype (see FIG. 1).
The expression xe2x80x9crelevant part of a suitable HBV genexe2x80x9d refers to the part of the HBV gene encompassing the HBV genotype specific target sequence chosen from FIG. 1 to be detected.
According to a preferred embodiment of the present invention, step (iii) is performed using a set of at least 2, preferably at least 3, more preferably at least 4 and most preferably at least 5 probes all meticulously designed such that they show the desired hybridization results, when used in a reverse hybridisation assay format, more particularly under the same hybridization and wash conditions implying that each of said probes is able to form a complex upon hybridisation with its target sequence present in the polynucleic acids of the sample as obtained after step (i) or (ii).
The numbering of the HBV gene encoded amino acids and nucleotides is as generally accepted in literature.
More particularly, the present invention relates to a set of at least 2 probes allowing the detection of a genotype specific variation, possibly also including one or more probes allowing the detection of a wild-type sequence, a polymorphic or a mutated sequence at any one of the nucleotide positions showing a sequence diversity upon alignment of all known or yet to be discovered HBV sequences as represented in FIG. 1 for all complete HBV genomes found in the EMBL/NCBI/DDBJ/Genbank.
The sets of probes according to the present invention have as a common characteristic that all the probes in said set are designed so that they can be used together in a reverse-hybridization assay, more particularly under similar or identical hybridization and wash conditions as indicated above and below.
Selected sets of probes according to the present invention include probes which allow to differentiate any of the HBV genotype specific nucleotide changes as represented in FIG. 1, preferably in the preS1 or HBsAg region of HBV. Said probes being characterized in that they can function in a method as set out above.
In order to solve the above-mentioned problem of obtaining information or, the possible presence of HBV mutants in a given sample, the present invention provides an elegant way to tackle this problem which involves residing to a reverse hybridisation approach (particularly on Line Probe Assays strips, as described by Stuyver et al., 1993). Using this technology it is possible to conveniently obtain all essential data in one test run. To achieve this goal, a set of probes needs to be designed and assembled which can detect all relevant mutations and possibly also wild-type sequences or polymorphisms in the HBV gene regions of interest.
Another particularly preferred embodiment of the present invention thus is a method for determining the presence or absence of one or more HBV mutants in a biological sample, comprising:
(i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample;
(ii) if need be amplifying the relevant part of a suitable HBV gene present in said sample with at least one suitable primer pair;
(iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two nucleotide probes hybridizing specifically to a HBV mutant target sequence chosen from FIG. 1, with said probes being applied to known locations on a solid support and with said probes being capable of hybridizing to the polynucleic acids of step (i) or (ii) under the same hybridization and wash conditions, or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T of said target sequence is replaced by U and with said set or probes possibly also comprising one or more wild-type HBV probes corresponding with the respective mutated HBV target sequence;
(iv) detecting the hybrids formed in step (iii);
(v) inferring the HBV mutant(s) present in said sample from the differential hybridization signal(s) obtained in step (iv).
It is to be understood that the term xe2x80x9cmutant target sequencexe2x80x9d not only covers the sequence containing a mutation, but also the corresponding wild-type sequence. The HBV mutant target sequence according to the present invention can be any sequence including a HBV mutated codon known in the art or yet to be discovered. Particularly preferred HBV mutant target regions are set out below.
In order to solve the problem as referred to above of obtaining information on the essential issues for proper diagnosis of HBV (namely genotype and different mutations particularly mutations in the preCore region, vaccine escape mutations and RT gene mutations selected by treatment with drugs such as lamivudine and penciclovir), the present invention provides a particularly elegant way to obtain such complex information.
Moreover, careful analysis of the data obtained by the present inventors clearly revealed that combining the information concerning the preCore and escape mutants with data on the genotype is essential to allow adequate interpretation of the results. Hence it is highly advantageous to be able to produce all relevant data simultaneously.
In this method for diagnosing HBV mutants, preferably in combination with HBV genotyping, a set of probes selected as defined above may be used, wherein said set of probes is characterized as being chosen such that for a given HBV mutation, the following probes are included in said set.
at least one probe for detecting the presence of the mutated nucleotide(s) at said position;
at least one probe for detecting the presence of the wild-type nucleotide(s) at said position;
possibly also (an) additional probe(s) for detecting wild-type polymorphisms at positions surrounding the mutation position.
Inclusion of the latter two types of probes greatly contributes to increasing the sensitivity of said assays as demonstrated in the examples section.
Selected sets of probes according to the present invention include at least one probe, preferably at least two probes, characterizing the presence of a HBV mutation at nucleotide positions chosen from the preCore region of HBV, more particularly from the following list of codons susceptible to mutations in the HBV preCore region, such as codon 15 in genotype A, and for all genotypes: codon 28, codon 29, and codon 28 and 29, or in the preCore promoter region (see FIG. 1).
Said probes being characterized in that they can function in a method as set out above.
An additional embodiment of the present invention includes at least one probe, preferably at least two probes, characterizing the presence of a vaccine escape mutation in codon positions chosen from the HBsAg region of HBV, more particularly from the list of codons susceptible to mutations in the HBV HBsAg region, such as at codons 122, 126, 141, 143, 144 or 145 (see FIG. 1).
An additional embodiment of the present invention includes at least one probe, preferably at least two probes, characterizing the presence of a mutation in the RT pol gene region of HBV, that gives rise to resistance to drugs such as lamivudine and penciclovir, for instance mutation of M to V or to I at position 552 (in the YMDD motif), mutation of V to I at position 555, mutation of F to L at position 514, mutation of V to L at position 521, mutation of P to L at position 525 and mutation of L to M at position 528 (see FIG. 1).
In a selected embodiment, a combination of at least two oligonucleotide probes is used and said combination of probes hybridizes specifically to at least two of the following groups of target sequences:
a mutant target sequence chosen from the HBV RT pol gene region,
a mutant target sequence chosen from the HBV preCore region,
a mutant target sequence chosen from the HBsAg region of HBV,
a HBV genotype-specific target sequence.
For instance, an embodiment involves hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence chosen from FIG. 1 and at least one nucleotide probe hybridizing specifically to a HBV mutant target sequence chosen from FIG. 1.
Another selected embodiment involves, for instance, hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence chosen from FIG. 1 and at least one nucleotide probe hybridizing specifically to a HBV mutant target sequence chosen from the RT pol gene region as represented in FIG. 1.
Another selected embodiment involves, for instance, hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence chosen from FIG. 1 and at least one nucieotide probe hybridizing specifically to a HBV mutant target sequence chosen from the preCore region as represented in FIG. 1.
Another selected embodiment involves, for instance, hybridizing with at least one nucieotide probe hybridizing specifically to a genotype specific target sequence chosen from FIG. 1 and at least one nucleotide probe hybridizing specifically to a HBV vaccine escape mutant target sequence within the HBsAg region as represented in FIG. 1.
In a selected embodiment, a combination of at least three oligonucleotide probes is used and said combination of probes hybridizes specifically to at least three of the following groups of target sequences:
a mutant target sequence chosen from the HBV RT pol gene region,
a mutant target sequence chosen from the HBV preCore region,
a mutant target sequence chosen from the HBsAg region of HBV,
a HBV genotype-specific target sequence.
For instance, an embodiment involves hybridizing with at least one nucleotide probe hybridizing specifically to a genotype specific target sequence chosen from FIG. 1, and at least one nucleotide probe hybridizing specifically to a HBV mutant target sequence chosen from the preCore region as represented in FIG. 1, and at least one nucleotide probe hybridizing specifically to a HBV vaccine escape mutant target sequence chosen from the HBsAg region as represented in FIG. 1.
For instance, another embodiment involves hybridizing with at least one probe hybridizing specifically to a mutant target sequence from the HBV RT pol gene region of HBV, and at least one probe hybridizing specifically to a mutant target sequence from the HBsAg region of HBV, and at least one probe hybridizing specifically to a genotype-specific target sequence from the HBsAg region of HBV. According to this embodiment, the relevant part of the HBV genome can be amplified by use of one primer pair, for instance HBPr 75 and HBPr 94.
In a selected embodiment, a combination of at least four oligonucleotide probes is used and said combination of probes hybridizes specifically to all of the following groups of target sequences:
a mutant target sequence chosen from the HBV RT pol gene region,
a mutant target sequence chosen from the HBV preCore region,
a mutant target sequence chosen from the HBsAg region of HBV,
a HBV genotype-specific target sequence.
Particularly preferred embodiments of the invention thus include a set of probes as set out above comprising at least one, preferably at least two, at least three, at least four or more probe(s) for targeting one, preferably two, three or more nucieotide changes appearing in the alignment of HBV genomes as represented in FIG. 1.
Even more preferred selected sets of probes according to the present invention include probes derived from two of the same or different regions of HBV bearing HBV mutated nucleotides, or in addition also a third (set of) probe(s) characterizing the presence of a third HBV mutation at any of the positions shown in FIG. 1, or particular combinations thereof.
Particularly preferred is also a set of probes which allows simultaneous detection of HBV mutations at codons 15, 28 and 29 in the preCore region, possibly in combination with mutations in the preCore promoter regions, in combination with mutations at codons 122, 126, 141, 143, 144, 145 in the HBsAg region, possibly also in combination with mutations in the HBV pol gene at codons 514, 521, 525, 528, 552 or 555.
In the instances where the alignment of HBV genomes of FIG. 1 is referred to in this invention, it should be construed as referring to an alignment of all existing and future HBV genomes. The existing HBV genome sequences can be deduced from any database, such as the EMBL/NCBI/DDBJ/GENBANK database.
A preferred set of preCore, preS1, HBsAg and RT pol gene probes of this invention are the probes with SEQ ID NO 1 to 278 of Table 1 (see also FIG. 1).
Particularly preferred sets of probes in this respect are shown in FIG. 2 and in FIG. 4. The probes in FIG. 2 and in FIG. 4 were withheld after a first selection for preCore, preS1, HBsAg and RT pol probes.
The probes of the invention are designed for obtaining optimal performance under the same hybridization conditions so that they can be used in sets of at least 2 probes for simultaneous hybridization. This highly increases the usefulness of these Probes and results in a significant gain in time and labour. Evidently, when other hybridization conditions would be preferred, all probes should be adapted accordingly by adding or deleting a number of nucleotides at their extremities. It should be understood that these concomitant adaptations should give rise to essentially the same result, namely that the respective probes still hybridize specifically with the defined target. Such adaptations might also be necessary if the amplified material should be RNA in nature and not DNA as in the case for the NASBA system.
The selection of the preferred probes of the present invention is based on a reverse hybridization assay format using immobilized oligonucleotide probes present at distinct locations on a solid support. More particularly the selection of preferred probes of the present invention is based on the use of the Line Probe Assay (LiPA) principle which is a reverse hybridization assay using oligonucleotide probes immobilized as parallel lines on a solid support strip (Stuyver et al. 1993; international application WO 94/12670). This approach is particularly advantageous since it is fast and simple to perform. The reverse hybridization format and more particularly the LiPA approach has many practical advantages as compared to other DNA techniques or hybridization formats, especially when the use of a combination of probes is preferable or unavoidable to obtain the relevant information sought.
It is to be understood, however, that any other type of hybridization assay or format using any of the selected probes as described further in the invention, is also covered by the present invention.
The reverse hybridization approach implies that the probes are immobilized to certain locations on a solid support and that the target DNA is labelled in order to enable the detection of the hybrids formed.
The following definitions serve to illustrate the terms and expressions used in the present invention.
The term xe2x80x9cgenetic analysisxe2x80x9d refers to the study of the nucleotide_sequence of the genome of HBV by any appropriate technique.
The term xe2x80x9cHBV mutantxe2x80x9d refers to any HBV strain harbouring genomic variations with serological, genetical or clinical consequences.
The term xe2x80x9cvaccine escape mutantxe2x80x9d is reviewed in the introduction section and in example 7. The most important region lies between amino acid 124 and 147 of the HBsAg region, but can be extended from amino acid 114 to 150.
The term xe2x80x9cmutant resistant to drugs such as lamivudine and penciclovirxe2x80x9d is reviewed in the introduction section and in Example 8.
The term xe2x80x9cHBV genotypexe2x80x9d refers to HBV strains with an intergenotype variation of 8% or more based on a comparison of complete genomes.
The target material in the samples to be analyzed may either be DNA or RNA, e.g. genomic DNA, messenger RNA, viral RNA or amplified versions thereof. These molecules are also termed polynucleic acids.
It is possible to use genomic DNA or RNA molecules from samples susceptible of containing HBV in the methods according to the present invention.
Well-known extraction and purification procedures are available for the isolation of RNA or DNA from a sample (f.i. in Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press (1989)).
The term xe2x80x9cprobexe2x80x9d refers to single stranded sequence-specific oligonucleotides which have a sequence which is complementary to the target sequence to be detected.
The term xe2x80x9ctarget sequencexe2x80x9d as referred to in the present invention describes the nucieotide sequence of a part of wild-type, polymorphic or mutant HBV gene sequence to be specifically detected by a probe according to the present invention. The polymorphic sequence may encompass one or more polymorphic nucleotides; the mutant sequence may encompass one or more nucleotides that are different from the wild-type sequence. It is to be understood that the term xe2x80x9cmutant target sequencexe2x80x9d not only covers the sequence containing a mutation, but also the corresponding wild-type sequence. Target sequences may generally refer to single nucleotide positions, codon positions, nucleotides encoding amino acids or to sequences spanning any of the foregoing positions. In the present invention said target sequence often includes one, two or more variable nucleotide positions. In the present invention polynucleic acids detected by the probes of the invention will comprise the target sequence against which the probe is detected.
It is to be understood that the complement of said target sequence is also a suitable target sequence in some cases. The target sequences as defined in the present invention provide sequences which should at least be complementary to the central part of the probe which is designed to hybridize specifically to said target region. In most cases the target sequence is completely complementary to the sequence of the probe.
The term xe2x80x9ccomplementaryxe2x80x9d as used herein means that the sequence of the single stranded probe is exactly the (inverse) complement of the sequence of the single stranded target, with the target being further defined as the sequence where the mutation to be detected is located.
Since the current application requires the detection of single basepair mismatches, stringent conditions for hybridization are required, allowing in principle only hybridization of exactly complementary sequences. However, variations are possible in the length of the probes (see below). It should also be noted that, since the central part of the probe is essential for its hybridization characteristics, possible deviations of the probe sequence versus the target sequence may be allowable towards head and tail of the probe when longer probe sequences are used. These variations, which may be conceived from the common knowledge in the art, should however always be evaluated experimentally, in order to check if they result in equivalent hybridization characteristics as the exactly complementary probes.
Preferably, the probes of the invention are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. Particularly preferred lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridisation characteristics.
Probe sequences are represented throughout the specification as single stranded DNA oligonucleotides from the 5xe2x80x2 to the 3xe2x80x2 end. It is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U).
The probes according to the invention can be prepared by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-triester method.
The term xe2x80x9csolid supportxe2x80x9d can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip. Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH2 groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.
The term xe2x80x9clabelledxe2x80x9d refers to the use of labelled nucleic acids. Labelling may be carried out by the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labelled primers, or by any other method known to the person skilled in the art. The nature of the label may be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
The term xe2x80x9cprimerxe2x80x9d refers to a single stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength.
The expression xe2x80x9csuitable primer pairxe2x80x9d in this invention refers to a pair of primers allowing the amplification of part or all of the HBV gene for which probes are immobilized.
The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).
The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu and Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of Qxcex2 replicase (Lizardi et al. 1988; Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules known in the art.
The oligonucleotides used as primers or probes may also comprise nucleotide analogues such as phosphorothiates (Matsukura et al., 1987), alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984).
As most other variations or modifications introduced into the original DNA sequences of the invention these variations will necessitate adaptions with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However the eventual results of hybridisation will be essentially the same as those obtained with the unmodified oligonucleotides.
The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, etc.
The xe2x80x9csamplexe2x80x9d may be any biological material taken either directly from the infected human being (or animal), or after culturing (enrichment). Biological material may be e.g. expectorations of any kind, broncheolavages, blood, skin tissue, biopsies, sperm, lymphocyte blood culture material, colonies, liquid cultures, faecal samples, urine etc.
The sets of probes of the present invention will include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may be applied in two or more (possibly as many as there are probes) distinct and known positions on a solid substrate. Often it is preferable to apply two or more probes together in one and the same position of said solid support.
For designing probes with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied.
Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions, explained further herein, are known to those skilled in the art.
The stability of the [probe: target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and % GC result in a Tm about 2-10xc2x0 C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures.
Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5xc2x0 C. below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.
It is desirable to have probes which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid. In the present case, single base pair changes need to be detected, which requires conditions of very high stringency.
The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, preferred oligonucleotide probes of this invention are between about 5 to 50 (more particularly 10-25) bases in length and have a sufficient stretch in the sequence which is perfectly complementary to the target nucleic acid sequence.
Regions in the target DNA or RNA which are known to form strong internal structures inhibitory to hybridization are less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.
Standard hybridization and wash conditions are disclosed in the Materials and Methods section of the Examples. Other conditions are for instance 3xc3x97SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at 50xc2x0 C.
Other solutions (SSPE (Sodium saline phosphate EDTA), TMACI (Tetramethyl ammonium Chloride), etc.) and temperatures can also be used provided that the specificity and sensitivity of the probes is maintained. If need be, slight modifications of the probes in length or in sequence have to be carried out to maintain the specificity and sensitivity required under the given circumstances.
In a more preferential embodiment, the above-mentioned polynucleic acids from step (i) or (ii) are hybridized with at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of the above-mentioned target region specific probes, preferably with 5 or 6 probes, which, taken together, cover the xe2x80x9cmutation regionxe2x80x9d of the relevant HBV gene.
The term xe2x80x9cmutation regionxe2x80x9d means the region in the relevant HBV gene sequence where at least one mutation encoding a HBV mutant is located in a preferred parts of this mutation region is represented in FIG. 1.
Apart from mutation regions as defined above the HBV wild-type or mutant genomes may also show polymorphic nucleotide variations at positions other than those referred to as genotype specific or mutant specific variated positions as shown in FIG. 1.
Since some mutations may be more frequently occurring than others, e.g. in certain geographic areas or in specific circumstances (e.g. rather closed communities) it may be appropriate to screen only for specific mutations, using a selected set of probes as indicated above. This would result in a more simple test, which would cover the needs under certain circumstances.
In order to detect HBV genotypes and/or HBV mutants with the selected set of oligonucleotide probes, any hybridization method known in the art can be used (conventional dot-blot, Southern blot, sandwich, etc.).
However, in order to obtain fast and easy results if a multitude of probes are involved, a reverse hybridization format may be most convenient.
In a preferred embodiment the selected set of probes are immobilized to a solid support in known distinct locations (dots, lines or other figures). In another preferred embodiment the selected set of probes are immobilized to a membrane strip in a line fashion. Said probes may be immobilized individually or as mixtures to delineated locations on the solid support.
A specific and very user-friendly embodiment of the above-mentioned preferential method is the LiPA method, where the above-mentioned set of probes is immobilized in parallel lines on a membrane, as further described in the examples.
The invention also provides for a set of primers allowing amplification of the region of the respective HBV gene to be detected by means of probes. Examples of such primers of the invention are given in Table 1 and FIG. 1.
Primers may be labelled with a label of choice (e.g. biotine). Different primer-based target amplification systems may be used, and preferably PCR-amplification, as set out in the examples. Single-round or nested PCR may be used.
The invention also provides a kit for detection and/or genetic analysis of HBV genotypes and/or HBV mutants present in a biological sample comprising the following components:
(i) when appropriate, a means for releasing, isolating or concentrating the polynucleic acids present in said sample;
(ii) when appropriate, at least one suitable primer pair;
(iii) at least two of the probes as defined above, possibly fixed to a solid support;
(iv) a hybridization buffer, or components necessary for producing said buffer;
(v) a wash solution, or components necessary for producing said solution;
(vi) when appropriate, a means for detecting the hybrids resulting from the preceding hybridization.
(vii) when appropriate, a means for attaching said probe to a known location on solid support.
The term xe2x80x9chybridization bufferxe2x80x9d means a buffer enabling a hybridization reaction to occur between the probes and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions.
The term xe2x80x9cwash solutionxe2x80x9d means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
As illustrated in the Examples section, a line probe assay (LiPA) was designed for screening for HBV genotypes and/or HBV mutants. The principle of the assay is based on reverse hybridization of an amplified polynucleic acid fragment such as a biotinylated PCR fragment of the HBV gene onto short oligonucleotides. The latter hybrid can then, via a biotine-streptavidine coupling, be detected with a non-radioactive colour developing system.